Osteosarcoma is the most common malignancy of the bones, and although advances in chemotherapy and surgery had been achieved in recent years, the long-term survival rate has reached a plateau. The main reason for this is the aggressive malignant potential and poor response of the disease to chemotherapy. However, several studies have found that tumor resistance is associated with cancer stem cells (CSCs). To address this issue, in the present study, osteosarcoma cells were treated with specially designated concentrations of cisplatin (CDDP) in a mouse model. Hematoxylin and eosin staining analyses were performed to assess tissue structure, in vivo passaging and CDDP treatment. Drug resistance genes and well-established stemness genes were detected by quantitative polymerase chain reaction. A serum-starved sphere formation assay was adopted to evaluate the ability to generate spherical clones and flow cytometry as used to test the expression of the cluster of differentiation 117 and Stro-1 surface markers, known as markers of CSCs. It was found that CDDP could induce an effect of resistance in the osteosarcoma cells, which possessed cancer stem CSC properties, as shown by the elevated expression of CSC marker genes and the higher expression of the cluster of differentiation 117 and Stro-1 surface markers. Moreover, the cells that dissociated from the tumor tissues exhibited an increased ability to form sarcospheres. The results of this study provided a significant correlation between resistance and CSCs, and revealed a clue indicating that osteosarcoma recurrence is likely to be associated with CSCs.